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Supplementary Table 1 from Effect of Long-term Storage in TRIzol on Microarray-Based Gene Expression Profiling

Background: Although TRIzol is widely used for preservation and isolation of RNA, there is suspicion that prolonged sample storage in TRIzol may affect array-based gene expression profiling (GEP) through premature termination during reverse transcription.Methods: GEP on Illumina arrays compared paired aliquots (cryopreserved or stored in TRIzol) of primary samples of multiple myeloma (MM) and acute myeloid leukemia (AML). Data were analyzed at the “probe level” (a single consensus value) or “bead level” (multiple measurements provided by individual beads).Results: TRIzol storage does not affect standard probe-level comparisons between sample groups: different preservation methods did not generate differentially expressed probes (DEP) within MM or AML sample groups, or substantially affect the many DEPs distinguishing between these groups. Differences were found by gene set enrichment analysis, but these were dismissible because of instability with permutation of sample labels, unbalanced restriction to TRIzol aliquots, inconsistency between MM and AML groups, and lack of biological plausibility. Bead-level comparisons found many DEPs within sample pairs, but most (73%) were Conclusions: TRIzol preserves RNA quality well, without a deleterious effect on GEP. Samples stored frozen with and without TRIzol may be compared by GEP with only minor concern for systematic artifacts.Impact: The standard practice of prolonged sample storage in TRIzol is suitable for GEP. Cancer Epidemiol Biomarkers Prev; 19(10); 2445–52. ©2010 AACR.

Supplementary Table 2 from Effect of Long-term Storage in TRIzol on Microarray-Based Gene Expression Profiling

Supplementary Table 3 from Effect of Long-term Storage in TRIzol on Microarray-Based Gene Expression Profiling

Focusing on triangular gutters with one vertical side, four issues pertaining to the design of street gutters are addressed: (1) the proper coefficient in Manning’s formula for uniform flow and friction loss in gutters; (2) adjustment of frictional resistance for spatial inflow; (3) whether uniform flow occurs in gutters with spatially increasing flow; and (4) interaction with inlets. The first of these issues is addressed by considering historical and modern information to recommend the best of two different equations that have been put forward for determining the uniform flow capacity and friction loss in gutters. The second concern is addressed for subcritical and supercritical gutter flow, for which practical, generalized numerical solutions are derived. The range of parameters for which uniform flow provides an adequate approximation is demonstrated theoretically and by example. Among the important conclusions is that the common practice of using Manning’s equation alone for such problems is...

Acute myeloid leukemia (AML) is sustained by a population of cancer stem cells (CSCs or cancer-initiating cell). The mechanisms underlying switches from CSCs to non-CSCs in vivo remain to be understood. We address this issue in AML from the aspect of epigenetics using genome-wide screening for DNA methylation and selected histone modifications. We found no major differences in DNA methylation, especially in promoter CpG islands, between CSCs and non-CSCs. By contrast, we found thousands of genes that change H3K4me3 and/or H3K27me3 status between stem and progenitor cells as well as between progenitor and mature cells. Stem cell related pathways and proliferation or metabolism related pathways characterize genes differentially enriched for H3K4me3/H3K27me3 in stem and progenitor populations. Bivalent genes in stem cells are more plastic during differentiation and are more likely to lose H3K4me3 than to lose H3K27me3, consistent with increasingly closed chromatin state with differentiation. Our data indicates that histone modifications but not promoter DNA methylation are involved in switches from CSCs to non-CSCs in AML.

Dysregulation of cyclin D2 contributes to the pathogenesis of multiple myeloma, and can occur through translocations that activate MAF/MAFB or MMSET/FGFR3. However, cyclin D2 induction can also be seen in the absence of such translocations, such as in patients with hyperdiploid disease, through unknown mechanisms. In UniGene cluster data-mining and ECgene analysis, we found that zinc-finger with KRAB and SCAN domains 3 (ZKSCAN3), a novel transcription factor, is overrepresented in this malignancy, and three consensus ZKSCAN3 binding sites were found in the cyclin D2 promoter. Analysis of a panel of myeloma cell lines, primary patient samples and datasets from Oncomine and the Multiple Myeloma Genomics Portal (MMGP) revealed expression of ZKSCAN3 messenger RNA (mRNA) in a majority of samples. Studies of cell lines by western blotting, and of primary tissue microarrays by immunohistochemistry, showed ZKSCAN3 protein expression in a majority, and in a manner that paralleled messenger levels in cell lines. ZKSCAN3 overexpression was associated with increased gene copy number or genomic DNA gain/amplification in a subset based on analysis of data from the MMGP, and from fluorescence in situ hybridization studies of cell lines and primary samples. Overexpression of ZKSCAN3 induced cyclin D2 promoter activity in a MAF/MAFB-independent manner, and to an extent that was influenced by the number of consensus ZKSCAN3 binding sites. Moreover, ZKSCAN3 protein expression correlated with cyclin D2 levels in cell lines and primary samples, and its overexpression induced cyclin D2. Conversely, ZKSCAN3 suppression using small hairpin RNAs (shRNAs) reduced cyclin D2 levels, and, importantly, inhibited myeloma cell line proliferation. Finally, ZKSCAN3 was noted to specifically bind to oligonucleotides representing sequences from the cyclin D2 promoter, and to the endogenous promoter itself in myeloma cells. Taken together, the data support the conclusion that ZKSCAN3 induction represents a mechanism by which myeloma cells can induce cyclin D2 dysregulation, and contribute to disease pathogenesis.

The present paper presents a generalized solution for the hydrologic and hydraulic design of small to medium-sized storm-water pumping stations. The solution enables determination of pump capacity and the corresponding storage capacity. The benefits of the solution include: (1) the ability to consider the effects of storm duration rather than the fixed hydrology assumed in other methods; (2) a simpler and more intuitive design procedure than other available methods; and (3) enabling the calculation of storage requirements independent of the geometry of the wet well and any inundated upstream area. The relation of the method presented herein to several other available methods is presented and the other methods critiqued conceptually and by example. The example is favorably tested using commercial software, and the constant-pumped-discharge assumption of the example is further tested using that software for an actual pump discharge-total dynamic head and system flow-head relationship. Extension of the metho...

Background: Although TRIzol is widely used for preservation and isolation of RNA, there is suspicion that prolonged sample storage in TRIzol may affect array-based gene expression profiling (GEP) through premature termination during reverse transcription. Methods: GEP on Illumina arrays compared paired aliquots (cryopreserved or stored in TRIzol) of primary samples of multiple myeloma (MM) and acute myeloid leukemia (AML). Data were analyzed at the “probe level” (a single consensus value) or “bead level” (multiple measurements provided by individual beads). Results: TRIzol storage does not affect standard probe-level comparisons between sample groups: different preservation methods did not generate differentially expressed probes (DEP) within MM or AML sample groups, or substantially affect the many DEPs distinguishing between these groups. Differences were found by gene set enrichment analysis, but these were dismissible because of instability with permutation of sample labels, unbalanced restriction to TRIzol aliquots, inconsistency between MM and AML groups, and lack of biological plausibility. Bead-level comparisons found many DEPs within sample pairs, but most (73%) were Conclusions: TRIzol preserves RNA quality well, without a deleterious effect on GEP. Samples stored frozen with and without TRIzol may be compared by GEP with only minor concern for systematic artifacts. Impact: The standard practice of prolonged sample storage in TRIzol is suitable for GEP. Cancer Epidemiol Biomarkers Prev; 19(10); 2445–52. ©2010 AACR.

Manifolds are used extensively in pipeline engineering applications. They may be classified as combining-flow in which the flow rate increases along the length of the manifold, and dividing-flow in which the flow rate decreases along the manifold length. The present paper addresses dividing-flow manifolds with the emphasis on pressure-distribution systems. The seminal theoretical and experimental work of McNown on discrete tee takeoffs, applicable to pressure-distribution-system manifolds, is discussed and elaborated upon for flow across the run of the tee and into the tee branch. Pertinent theoretical and experimental work for discrete orifice takeoffs is summarized. The information thus presented facilitates discussion of certain concepts presently incorporated in pressure-distribution-system design. Certain erroneous concepts are identified. A perspective is provided and improvements in related aspects of manifold design are suggested. A separate paper, presented at the ASCE/EWRI World Environmental an...

Basic relationships are derived for calculating the flow attenuation and depth of storm water on roofs. Hyetographs and corresponding hydrographs are specialized to the case of roof drainage and incorporated in methods which are presented for analysis and design of roof drains. Roof storage and outflow are characterized for various roof geometries and outflow devices. The methods are applied to examples found in the literature, including those in standards of Factory Mutual Insurance Company, ASCE, and others. Assumptions implicit in those standards are tested and their limitations demonstrated. A critical duration at which the greatest water depth and outflow occur must be found on a case-by-case basis. The critical durations can differ significantly from the fixed durations specified in standards. The corresponding maximum water depths and peak outflows can also differ accordingly. For determination of roof loads, these results will have their greatest importance in warmer climates in which the rain loads govern over snow loads. An example of design specifically for flow attenuation is also presented.

A particularly useful generalized numerical solution for detention basin design is presented and sets the stage for practical applications for roof detention and the rationale and use of such a solution for detention basin design more generally. The generalized numerical solution provides results that can be applied to practical problems in certain cases and demonstrates the general importance of storm duration. The method utilizes a trapezoidal inflow hydrograph, which includes the important effect of storm duration based on the modified rational method. Convenient charts are presented to display the relationship between the variables. The derivation provides the basis for specializing the method to provide generalized curves applicable to storage detention on roofs. Although the results presented herein are more generalized than those presented by other writers, certain other methods are shown to compare favorably for special cases. An added perspective is provided on some of those other methods. Soil Conservation Service TR-55 curves are included in the comparisons, which demonstrates their limitations. An example demonstrates application of the generalized curves.

The hydraulic design of certain types of subsurface drains has recently been put on a more rational footing, and deficiencies in earlier design methods have been demonstrated. However, significant limitations remain in hydraulic design methods for geosynthetic and aggregate subsurface drains. It is important to decouple the groundwater hydrology from the internal hydraulics of the drain, and properly design subsurface drains for open-channel rather than pressurized conditions. Present design methods can inadvertently result in pressurized flow. The assumption of uniform flow (Manning’s equation alone) is also improperly made in some present design methods. Consequences can include unintended pressurized flow and attendant nonuniformity of inflow on the one hand and uneconomical design on the other. Current standard guidelines provide relatively little guidance for the design of geosynthetic and aggregate drains. A current ASTM standard, commonly referenced by geosynthetic manufacturers, has significant li...

Collection conduits flowing full with nonuniform inflow (variable rate of increase in flow with distance along the conduit length) include well screens (vertical and directionally drilled); submerged effluent collectors; and certain types of inboard weir configurations for settling tanks. Certain subsurface drains used in environmental engineering applications and civil engineering more generally may be inadvertently designed for full-flowing conditions. Formulation of the problem for such collection conduits is presented in terms of the applicable differential equation, slope invariance, the frictionless solution for a general cross section, a uniform-inflow solution, and the difference formulation. The importance of checking inflow uniformity is discussed and exemplified. Dimensional analysis is then employed to demonstrate the relationship between variables, leading to a new generalized numerical solution. That solution is presented in a graphical form which provides further useful display of the relationships between variables and quantitative information for design and analysis. The detrimental effects of flowing-full conditions in subsurface drains is demonstrated, and it is noted that existing design methods may unintentionally cause such conditions to occur.

A theory presented previously by the writer explained the asymmetric flow patterns occurring in symmetric rectangular expansions with subcritical flow. The theory is extended in the present paper to supercritical flows, in which the hydraulic jump is one of the flow features. The theory is shown to agree with available observations. Transverse oscillatory conditions are discussed in terms of a dynamic instability that can accompany static stability. The ranges for which experimental data are available with which to test the theory are identified, making it apparent where additional data would be useful for the full range of the problem. The phenomenological understanding provided by the present paper is important to the development of stabilization measures. In addition to stabilization by submergence, the hydraulic jump should be amenable to stabilization by other referenced methods, including various vane configurations and pipe communication between the eddy regions.

Numerous types of pipes and channels with spatially increasing flows in environmental engineering applications are identified by type and function and referred to as collection conduits. An overview of methods for designing and analyzing collection conduits is provided. Full conduits with nonuniform and uniform inflow are first considered. Dimensional analysis is then employed to demonstrate the relationship between variables for open channels; that leads to the identification of possibilities for generalized numerical solutions. Prior collection conduit applications are discussed within the framework of the dimensional analysis (which also pertains to some constant-flow applications). A previously unpublished generalized numerical solution for rectangular collection conduits is presented. Subsurface drains are addressed with particular emphasis, including the use of numerical methods to develop a new generalized chart and relation to other design methods. Among the important conclusions for subsurface drains is that the somewhat common practice of using Manning’s equation alone for such problems is not generally adequate. Examples and practical design suggestions are included, and the use of computer-based numerical methods is discussed more generally.

A model has been developed and successfully applied to analyze a network of tidal ponds connected by culverts and bridges. Measured tidal fluctuations under normal and spring tide conditions at the seaward terminus of the tidal ponds were applied as boundary conditions. The model uses the secant modification of the Newton-Raphson method for solving a set of nonlinear differential equations. It was necessary to develop special conduit file generation and interpolation techniques to render the model feasible in terms of required computational time. The model adequately represented tidal fluctuations measured in the tidal ponds under existing conditions. The model was then applied to predict changes in tidal fluctuations resulting from replacement of one of the culverts with alternative bridge designs with and without freshwater runoff. The predictions enabled separate assessment of impacts on shore-side properties with individual septic systems and on water quality, flora, and fauna within the tidal ponds.

Analysis of a network of tidal ponds connected by hydraulically complex conduits requires special conduit file generation and interpolation techniques. Hydraulically complex in this context means that computer-intensive calculations are required to adequately determine flow through each connecting conduit for each combination of headwater and tailwater elevations (e.g., for unsubmerged culverts). Such file generation and interpolation techniques were developed for a specific application that enabled the successful analysis of a network of tidal ponds connected by culverts and bridges. The techniques decreased computational times of an otherwise optimized program by orders of magnitude. Without such techniques, such modeling would not be feasible in terms of required computational time. The computer run times (Pentium-133) of the final model runs ranged from about 10 h or 8.8 times real time to about 3 days or 0.67 times real time, depending on the tide conditions considered and the hydraulic complexity of...

Building upon a companion paper, the present paper addresses additional mechanisms of the tidal bore, including channel taper and friction; building, maximum height, variation, and dissipation of the bore; and standing waves. Early studies of these mechanisms are cited and discussed in historical context. Relationships developed to investigate the effects of horizontal channel taper, vertical channel taper, and channel friction agree qualitatively with and explain field observations. Building of the bore is explained, with favorable comparison of methodology with flume observations. The maximum height that the bore is capable of achieving is one of its most interesting features; this is explained in terms agreeing with experimental observation. The two forms of the jump, undular and wall-like, are discussed. The variation of the bore height as it moves into regions of greater or lesser depth is described from field observations and flume observation, and relationships derived which explain such variation. Frictional dissipation of the bore is discussed. Standing waves that occur after the tidal bore has passed are explained as a separate phenomenon.Copyright © 2012 by ASME

Tidal bores have been the subject or cause of legend, poetry, recreation, tourism, shipping disasters, and scientific interest for centuries. They pose interesting theoretical questions for the engineer. Introductory details are presented regarding the remarkable tidal bore phenomenon, including discussion of the tidal bore in various locations and a recent photograph taken by the writer of the bore on China’s Qiantang River. A supplemental tabular summary is presented based on an extensive literature review. A simplified theory based on monoclinal rising waves is developed for the initiation of the tidal bore. The theory elucidates the mechanisms of the tidal bore more clearly and completely than more complex theories. Early studies of the tidal bore are cited and discussed in historical context. The intention is to provide greater understanding of the tidal bore for a broad set of readers. The theory is favorably compared to rectangular flume data and to all field data available to the writer. A companion paper builds on the present one by addressing additional features of the tidal bore.Copyright © 2012 by ASME

By properly considering well-established concepts of theoretical and experimental fluid mechanics, inadequacies of methods in widespread use for wastewater pressure-distribution system design are revealed. Erroneous concepts include deficient consideration of pressure changes and velocity-head recovery in the manifold and lateral across takeoffs, and oversimplified, inaccurate consideration of the head loss in the branch (riser) entrance. Improvements are presented that can result in correct design with better performance and economy. For design purpose it is useful to have a simplified method for selecting design parameters for laterals and manifolds and a computer-based method to check and refine the design; that is the approach utilized by the writer and to be elaborated upon in a future paper. A larger perspective is also presented, by noting numerous other concepts of "established practice" that the author has overturned and for which he has provided corrective methods. This is offered as encouragement to others to not simply accept published design methods, but rather to critically review their validity and applicability and modify them for specific applications and more generally when appropriate.

The control of pain and the alleviation of anxiety are integral components in the management of soft tissue injuries in the Emergency Department. Pharmacologic agents utilized for analgesia and sedation must possess properties favorable for emergency department use. This article reviews the pharmacologic approach to patients who require sedation and/or analgesia for the management of a soft tissue injury in the Emergency Department.

The relationship for combined increasing and decreasing free surface flow is presented in difference form, and then particularized to spatially increasing and spatially decreasing flows. For spatially decreasing flow, a theoretical difference is noted between the writer’s version and that in a widely-used reference source. By investigating an application for uniformly decreasing, full-pipe flow, it is shown by physical reasoning and comparison of an available analytical solution to the alternate versions of the difference relationship that the writer’s difference relationship is the correct one. Free surface spatially varied flow relationships are specialized to pressure conduits to demonstrate conditions of slope invariance, which has important and somewhat counter-intuitive ramifications for certain practical applications encountered in environmental engineering.Copyright © 2004 by ASME

By presenting relationships for spatially varied flow with combined increasing and decreasing discharge, and relating those relationships to prior work, a clearer exposition of spatially varied flow concepts is provided. The applicability and usefulness of energy (First Law of Thermodynamics) and momentum (Newton’s Second Law) principles to spatially varied flows are addressed. The history of the debate initiated in the nineteenth century over the role of the First Law of Thermodynamics in general and hydrodynamics specifically is discussed, related to current issues regarding spatially varied flows, and hopefully resolved in the context of such flows and more generally.Copyright © 2004 by ASME

) and could drive them to extinction ifmitigating measures are not taken. But in fact,some of the underlying factors in this modeldo not represent actual conditions.Eagles are protected under the Bald andGolden Eagle Protection Act and theEndangered Species Act. In March,the fox subspecies on each ofthe northern Channel Islandswere listed as endangered,and measures to preventfox extinction and ulti-mately provide forrecovery are being taken.Between 1999 and 2002,foxes were captured andbrought into captivity on allthree of the northern ChannelIslands, to be held until the threatfrom eagles is further reducedor eliminated, and toincrease wild fox popula-tions through captivebreeding and release. Aworking group of 90professionals advisesthe fox recovery effort.Since 1999, 35 goldeneagles have beencaptured and relocated tonorthern California.Despite employment of themost effective known goldeneagle capture techniques, someeagles evade capture and continue tobreed and prey on foxes.Running a captive breeding program onthree island locations is not without its ownrisks, particularly from disease, loss of geneticvariation, and changes in behavior. For thosereasons, and to learn more about the efficacyof restoration in the face of a novel predator,foxes were released from the breeding facili-ties on Santa Cruz and Santa Rosa Islandsstarting in December 2003. On Santa Cruz,five of the nine foxes released were killed bygolden eagles, and the remaining four werereturned to captivity. On Santa Rosa, one ofthe released foxes died of eagle predation,seven remain in the wild, and a pair of thereleased foxes has produced two pups.Captive-bred foxes seem much more suscep-tible to eagle predation. In contrast, annualsurvivorship of the remaining wild foxes onSanta Cruz was 80% in 2003, as determinedby radiotelemetry.Even with a high population of pigspresent, the island foxes released fromcaptivity experienced a high predation rate,suggesting that they were the preferred foodfor some eagles or the more accessible food insome areas. Moreover, the removal of the pigson Santa Cruz is necessary for the recovery ofnine endangered or threatened plants. Baldeagles (

Abstract 3640 Background & Aims: The hypothesis that cancer is driven by Cancer Stem Cells (CSCs or Cancer-Initiating Cell) has recently attracted a great deal of attention. Epigenetic mechanism such as DNA methylation and histone modification play an important role in cancer cells and also in normal stem cells. However, their role remains unclear in CSCs. We sought to determine if CSCs have distinct epigenetic patterns in acute myeloid leukemia (AML). Methods: Peripheral blood samples in AML patients were separated to obtain stem cells (CD34+CD38-) and progenitor cells (CD34+CD38+) by magnetic cell sorting (MACS®, Myltenyi biotec). To study DNA methylation in CSCs in AML, we performed genome wide screening using methylated CpG island microarray (MCAM), which detects 7202 promoter CpG islands, 1348 non-promoter CpG islands, and 632 non-CpG island promoter methylation. MCAM was performed on 4 AML patient samples Next, we evaluated the methylation status of 7 genes which showed apparent higher DNA methylation in stem cells or progenitor cells in MCAM analysis, using a quantitative bisulfite-pyrosequencing for each population of stem cell, progenitor cell, and mature cells (CD34-) from peripheral blood samples in 6 AML patients. For histone modification analysis, we used Chromation immuprecipitation followed by massively parallel sequencing (ChIP-Seq) for stem cell and progenitor cell populations for H3K4me3 which is known to be a marker for activated genes. Results: By MCAM, we found minimal differences between stem cells and progenitor cells present in 2 out of 4 AML patients. Those few genes ( Conclusions: There is no significant difference in DNA methylation between stem cell, progenitor cell or mature cell populations in AML. DNA methylation of promoter CpG islands is unlikely to explain tumor hierarchy in AML. Rather, histone modifications seem to have a greater significance in this regard. Disclosures: No relevant conflicts of interest to declare.

Abstract 2126 Background. Studying the cellular biology of myelodysplasia (MDS) has been greatly hindered by limitations in the amount of material that can be obtained from patients and by fact that the neoplastic MDS cells are a small fraction of the total bone marrow population. Sample requirements for Reverse Phase Protein Array (RPPA) are small enough to enable array like analyses that were previously impossible. We therefore constructed an RPPA to perform proteomic profiling in MDS that analyzed both the CD34+ and the stem cell enriched CD34+CD38- fractions. Methodology. Fresh (F) or cryopreserved (C) primary bone marrow (BM, n=153 F and 51C) or blood (PB n=84 F and 7 C) samples from 185 MDS patients (123 diagnosis, 62 relapse) underwent sequential CD34 selection followed by CD38 depletion using magnetic antibody sorting to produce CD34+ and CD34+CD38- (SC) fractions. All samples yielded sufficient CD34+ cells, with a median yield of 4×105 cells and recovery of 3.2%. CD34+CD38- recovery correlated with the starting number of cells and was successful 50% of the time when starting with >1×107 cells, with a median yield of 3×105 cells and recovery of ∼ 2% of the starting number of cells. There were 91 Paired CD34+/SC samples from 82 cases and 27 paired BM/PB and 16 paired DX/Rel samples. Slides were probed with 128 antibodies (ABs) against apoptosis, cell cycle, signaling (STP), regulating proteins, integrins, phosphatases etc including 90 vs. total protein, 33 vs. phos-specific sites and 5 vs. caspase or PARP cleavage sites. Spot intensities were quantified using MicroVigene software. Data was analysed using R, with loading control and topographical background normalization being utilized. Results. A mixed linear model was used to compare protein expression in CD34+ vs. SC. Of the 128 proteins 82 were significantly different with a FDR of 5%. The majority of SC clustered separately from the CD34+ cells but there were a few SC with CD34+ like expression and about 25% of CD34+ cases were found in the SC group. We therefore looked at protein expression patterns in CD34+ and SC separately. Protein expression patterns distinguished FAB and IPSS groups for both CD34+ and SC, and with 8 and 11 proteins being significantly different among CD34 + cells and 15 and 8 being different in the SC. Proteins were clustered based on Pearson and complete linkage. In CD34+ cells, the Gap statistic suggested that there were 11 constellations of protein with correlated expression. Principle component analysis identified 6 distinct Protein Expression Signatures (ProExpSig) based on the expression of the 11 constellations. In the SC analysis there were 8 protein constellations and principle component analysis identified 4 distinct ProExpSig. Perturbation boostrap clustering of the protein expression constellations and the ProExpSig showed that they were highly reproducible for both the CD34+ and SC analyses. Signature assignment was more strongly associated with FAB in the SC than CD34+ analysis. In contrast IPSS score was more strongly associated with signature assignment. Concordance of ProExpSig between the CD34+ and SC groups was minimal reflecting the differences in protein expression. Among the patients that were treated is was obvious that there were favorable and unfavorable signatures. For the CD34+ analysis ProExpSig 1,2 and 3 were unfavorable and had a median survival of 71 weeks compared to favorable groups 4, 5, 6 which had a median survival of 127 weeks (p=0.009). A similar finding was present in the SC analysis where ProExpSig 1 and 2 were unfavorable with a median survival of 80 weeks compared to favorable ProExpSig 3 ad 4, which had a median survival of 128 weeks (p=0.08). When combined, the information from both the CD34+ and SC analyses improved the prognostic discrimination (P=0.007) with the four groups having median survival of 50, 53, 97 and 248 weeks. Conclusions. This is the first study to perform proteomic profiling in MDS or in MDS stem cells. Protein expression in SC was markedly different from CD34+ cells stressing the importance of studying this rare population to truly understand the biology of MDS. Similar to our findings in AML and ALL, MDS is characterized by several distinct ProExpSig which form favorable and unfavorable prognostic groups. The differences in the patterns of protein expression can be exploited therapeutically to target specific agents to those cases where that particular pathway or protein is active. Disclosures: Kantarjian: AstraZeneca: Research Funding.

We used paired samples to address whether long-term storage of cells in TRIzol has a significant effect on microarray-based gene expression profiling (GEP). The TRIzol reagent is widely used for the preservation and isolation of RNA, but there is suspicion that prolonged storage of tissues prior to RNA isolation, even at −80 C., can cause chemical modification (depurination) of RNA. This could result in early termination during reverse transcription (RT) of mRNA molecules, potentially affecting GEP more strongly for transcripts with probes located farther from the 3’ end. The Myeloma and Leukemia Tissue Banks at M. D. Anderson Cancer Center routinely collect and store primary tumor samples for research under informed consent. From these banks we selected primary tumor samples of multiple myeloma (MM; CD138+) and acute myeloid leukemia (AML; Ficoll-purified, CD3- and CD19-depleted), for which paired aliquots had been stored frozen in TRIzol (“Tri”) or cryopreservation medium (RPMI + 20% FCS + 10% DMSO, “Cryo”) since the time of initial isolation (range, 2-9 years). Cryo aliquots were quickly thawed, washed, and placed in TRIzol, then total RNA was isolated from all aliquots as per the TRIzol manufacturer's instructions. RNA quality was assessed with a Bioanalyzer 2100 (Agilent); the RNA integrity number (RIN) was > 9 for 12/12 AML Tri, 6/12 AML Cryo, 6/6 MM Tri, and 3/6 MM Cryo samples. RNA from 12 sample pairs (6 AML, 6 MM, all with RIN > 7) was further purified with Qiagen RNAeasy columns, and the Illumina® TotalPrep RNA Amplification Kit (Ambion) was used to generate amplified, biotinylated cRNA after RT by the Eberwine procedure. Bioanalyzer-generated histograms of cRNA were similar for all sample aliquots, suggesting no early termination of RT due to TRIzol. GEP was performed with cRNA on Illumina HT-12 BeadArrays. Analysis of “probe-level” data with GenomeStudio software (Illumina), after median normalization, showed excellent concordance between sample Tri/Cryo pairs for all but probes with low intensities ( 13 for any sample pair, and most pairs had Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2177.

The majority of studies of Acute Myelogenous Leukemia (AML) cell biology that are performed on patient derived samples utilize the more readily available bulk mononuclear leukemia cells (BULK) instead of rare stem cells (SC), defined as CD34+CD38− for this analysis. However, resistance of SC to therapy is thought to be more clinically relevant with SC leading to relapse and mortality. If the cellular machinery within the SC compartment is significantly different from that of the Bulk cells then these studies could generate results of uncertain veracity arising from studying the wrong cell. The ability to measure gene and protein expression in SC would answer this question but to date conventional methodology has required too many cells to permit the analysis of protein expression and activation patterns within SC. Using RPPA methodology on blood (PB) or marrow (BM) derived AML cells we have previously demonstrated that recurrent protein expression signatures with prognostic implications exist in AML. Our RPPA methodology requires only 333 cells per slide making the analysis of protein expression in stem cells possible for the first time. We therefore set out to determine whether protein expression in AML SC differed from BULK cells. Leukemia enriched (CD3/CD19 depleted) mononuclear cell fractions from 103 AML samples from 87 patients (including 13 paired PB and BM and 5 paired diagnosis and relapse samples) were separated in to CD34− and CD34+ fractions, followed by separation of the CD34+ fraction into CD34+CD38+ and CD34+CD38− fractions using magnetic antibody selection. Among the 103 samples 8 had only CD34+/− samples, 31 had BULK/CD34+/CD34−, 18 had BULK/CD34+/CD34−/CD34+CD38+, and 45 had all 5 samples. Whole cell lysates and total RNA preparations were made from these fractions. RPPA were printed using 5 serial 1:2 dilutions, printed in replicate. Slides were stained with 127 antibodies (predominantly apoptosis, cell cycle and STP regulating proteins) detecting 87 total, 37 phopho and 3 caspase cleavage sites. Spot intensities were quantified using MicroVigene software. Data was analyzed using R, with loading control and topographical background normalization being utilized. Analysis was performed using a mixed-effects model and β-uniform model. We compared expression between the BULK, and SC fractions, between CD34+ and CD34− fractions and between CD34+CD38+ and C34+CD38−(SC) fractions. In univariate analysis, protein expression in SC was higher for Caspase8, ZNF342, α-Catenin, and TRIM24, and lower for, Bax, pP70S6K and pS6RP across the entire population when compared to protein from BULK leukemic cells. The fold differences were subtle ranging from 15 to 27% increases and 13 to 38% decreases in expression in the SC relative to the BULK cells. There was no statistically significant evidence for global differences in individual protein expression for the CD34+/CD34− or CD34+CD38+/CD34+CD38− comparisons. In multivariate analysis, when patterns of expression of all 127 proteins were compared, the majority of samples from individual patients showed close correlation regardless of whether BULK/SC, CD34+/CD34− or CD34+CD38+/CD34+CD38− were compared. Gene expression profiling of these same samples using mRNA amplification techniques is underway. Comparison of differences in GEP and RPPA will be performed at a later date. In summary, the RPPA technique has enabled the analysis of expression of numerous proteins and their activation state in AML stem cells for the first time. We observed that protein expression in SC generally is similar to that of BULK cells. This increases confidence in the ability to generalize the results obtained in studies of bulk AML cells. A few proteins did show consistent differences in expression between stem and bulk cells. Novel agents targeting these should be investigated for the ability to individually or synergistically target AML SC.

The Pursuit of Certainty: Religious and Cultural Formulations. Wendy James, ed. London: Routledge Press, 1995. xi + 308 pp., index.

Asymmetric flow patterns may occur in perfectly symmetric abrupt expansions, in which the main flow deflects and attaches arbitrarily to one wall of the expansion. With the notable exception of “fluidic” devices, asymmetric flow patterns are detrimental in most civil engineering applications. The adverse effects include lengthening of the distance to full-width flow in expansions, inefficient operation of screens in screen channels, and increasing the potential for vortexing in pump intakes. No good explanation or prediction of the onset of asymmetric flow patterns is known to exist. This paper attributes the asymmetric behavior to a static instability of the system occurring under certain conditions of expansion geometry. The stability is investigated by analyzing the forces acting on the system when it undergoes a small deflection from its symmetric position. A mathematical stability criterion is developed and applied to subcritical, horizontal, rectangular expansions. The results are in excellent agreement with experimental observations (limited to small Froude Numbers). The predictive method is extended theoretically to the analysis of certain preventive and corrective measures. Means of extending the analyses to more complex two-dimensional and three-dimensional configurations are examined.